In myocardial tissue depolarization of the cells induces a transmembrane influx of Ca ions which triggers the contraction of myofibrils. A number of studies have identified unique characteristics of the voltage dependent slow Ca channels. The Ca antagonists, a class of negative inotropic coronary vasodilating drugs, are potent inhibitors of this influx system. Compelling electrophysiolgical evidence indiates that Ca channels must be phosphorylated to become activated in a voltage dependent manner (Sperelakis and Schneider, 1976). The goal of this research program will be to characterize the slow Ca channel in mammalian myocardial membranes. Biochemical assays for the Ca influx system will be developed by exploiting two properties: (1) cyclic antagonists such as verapamil or nifedipine are potent inhibitors of slow Ca influx. Sarcolemmal membranes from guinea pig and chick embryo hearts will be prepared. The substrates present in these preparations for endogenous cyclic AMP dependent protein kinase will be determined using SDS gel electrophoresis to separate 32p-labeled proteins. in subsequent studies microgram quantities of these proteins will be separated using preparative scale SDS polyacrylamide electrophoresis and/or isoelectric focusing. The strategy will be to generate specific antibodies directed against these substrates by injection into rabbits and then to screen these antisera for activity against the slow Ca channels in myocardial cells. In another approach, radioactive deriavatives of varapamil and nifedipine will be prepared in an attempt to develop radioligands which would be useful in a receptor binding assay. The goal of these studies will be to characterize the specific receptros for the Ca antagonist on sarcolemmal membranes. The chemical methods developed can then be applied to the synthesis of radioactive photoaffinity labels for these receptors. Covalent attachment of these ligands to the receptor will allow for their identification by SDS gel electrophoresis or other methods. It is the intent that the development of different markers or labels will provide a basis for further experiments designed to identify different functional components of the voltage dependent slow Ca influx system.